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Histone deacetylase 8 is deregulated in urothelial cancer but not a target for efficient treatment.

Lehmann M, Hoffmann MJ, Koch A, Ulrich SM, Schulz WA, Niegisch G - J. Exp. Clin. Cancer Res. (2014)

Bottom Line: Efficient siRNA-mediated knockdown of HDAC8 reduced proliferation up to 45%.Expression of thymidylate synthase was partly reduced; PARP-cleavage was not detected.The influence of the pharmacological inhibition on clonogenic growth and migration show a cell line- and inhibitor-dependent reduction with the strongest effects after treatment with compound 5 and compound 6.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: Previous studies have shown that class-I histone deacetylase (HDAC) 8 mRNA is upregulated in urothelial cancer tissues and urothelial cancer cell lines compared to benign controls. Using urothelial cancer cell lines we evaluated whether specific targeting of HDAC8 might be a therapeutic option in bladder cancer treatment.

Methods: We conducted siRNA-mediated knockdown and specific pharmacological inhibition of HDAC8 with the three different inhibitors compound 2, compound 5, and compound 6 in several urothelial carcinoma cell lines with distinct HDAC8 expression profiles. Levels of HDAC and marker proteins were determined by western blot analysis and mRNA levels were measured by quantitative real-time PCR. Cellular effects of HDAC8 suppression were analyzed by ATP assay, flow cytometry, colony forming assay and migration assay.

Results: Efficient siRNA-mediated knockdown of HDAC8 reduced proliferation up to 45%. The HDAC8 specific inhibitors compound 5 and compound 6 significantly reduced viability of all urothelial cancer cell lines (IC₅₀ 9 - 21 μM). Flow cytometry revealed only a slight increase in the sub-G1 fraction indicating a limited induction of apoptosis. Expression of thymidylate synthase was partly reduced; PARP-cleavage was not detected. The influence of the pharmacological inhibition on clonogenic growth and migration show a cell line- and inhibitor-dependent reduction with the strongest effects after treatment with compound 5 and compound 6.

Conclusions: Deregulation of HDAC8 is frequent in urothelial cancer, but neither specific pharmacological inhibition nor siRNA-mediated knockdown of HDAC8 impaired viability of urothelial cancer cell lines in a therapeutic useful manner. Accordingly, HDAC8 on its own is not a promising drug target in bladder cancer.

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Proliferation and clonogenicity in urothelial cancer cells after siRNA mediated knockdown of HDAC8. (A) Relative cell viability in several urothelial carcinoma cell lines after siRNA mediated knockdown of HDAC8 compared to irrelevant control (72 h). The percentage of viable cells was measured by ATP-assay and is displayed on the y-axis. p < 0.01 and p < 0.001 were defined as highly significant and marked as * and **. (B) Giemsa-staining of colonies from irrelevant siRNA and HDAC8 siRNA transfected RT-112, VM-CUB1, SW-1710, 639-V and UM-UC-3 cells compared to an untreated control (72 h).
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Figure 3: Proliferation and clonogenicity in urothelial cancer cells after siRNA mediated knockdown of HDAC8. (A) Relative cell viability in several urothelial carcinoma cell lines after siRNA mediated knockdown of HDAC8 compared to irrelevant control (72 h). The percentage of viable cells was measured by ATP-assay and is displayed on the y-axis. p < 0.01 and p < 0.001 were defined as highly significant and marked as * and **. (B) Giemsa-staining of colonies from irrelevant siRNA and HDAC8 siRNA transfected RT-112, VM-CUB1, SW-1710, 639-V and UM-UC-3 cells compared to an untreated control (72 h).

Mentions: To investigate the impact of HDAC8 on cell proliferation of UCCs we performed viability assays after 72 h of transfection. Targeting HDAC8 with siRNA caused a 20% to 45% reduction of cell growth compared to the irrelevant control (Figure 3A). Colony forming assays were performed to evaluate the role of HDAC8 for anchorage-dependent clonal growth capability. The siRNA mediated HDAC8 knockdown inhibited clonogenic growth of UCCs (Figure 3B). The transfection of HDAC8 siRNA in VM-CUB1 and UM-UC-3 cells caused a moderate reduction of colony numbers compared to transfection of irrelevant siRNA by up to 30%. The relative size of the HDAC8 siRNA transfected colonies is reduced in 639-V in comparison to irrelevant siRNA. In VM-CUB1, SW-1710, RT-112 and UM-UC-3 cells the colony size remains constant between irrelevant control and HDAC8 siRNA transfection (data not shown).


Histone deacetylase 8 is deregulated in urothelial cancer but not a target for efficient treatment.

Lehmann M, Hoffmann MJ, Koch A, Ulrich SM, Schulz WA, Niegisch G - J. Exp. Clin. Cancer Res. (2014)

Proliferation and clonogenicity in urothelial cancer cells after siRNA mediated knockdown of HDAC8. (A) Relative cell viability in several urothelial carcinoma cell lines after siRNA mediated knockdown of HDAC8 compared to irrelevant control (72 h). The percentage of viable cells was measured by ATP-assay and is displayed on the y-axis. p < 0.01 and p < 0.001 were defined as highly significant and marked as * and **. (B) Giemsa-staining of colonies from irrelevant siRNA and HDAC8 siRNA transfected RT-112, VM-CUB1, SW-1710, 639-V and UM-UC-3 cells compared to an untreated control (72 h).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4230422&req=5

Figure 3: Proliferation and clonogenicity in urothelial cancer cells after siRNA mediated knockdown of HDAC8. (A) Relative cell viability in several urothelial carcinoma cell lines after siRNA mediated knockdown of HDAC8 compared to irrelevant control (72 h). The percentage of viable cells was measured by ATP-assay and is displayed on the y-axis. p < 0.01 and p < 0.001 were defined as highly significant and marked as * and **. (B) Giemsa-staining of colonies from irrelevant siRNA and HDAC8 siRNA transfected RT-112, VM-CUB1, SW-1710, 639-V and UM-UC-3 cells compared to an untreated control (72 h).
Mentions: To investigate the impact of HDAC8 on cell proliferation of UCCs we performed viability assays after 72 h of transfection. Targeting HDAC8 with siRNA caused a 20% to 45% reduction of cell growth compared to the irrelevant control (Figure 3A). Colony forming assays were performed to evaluate the role of HDAC8 for anchorage-dependent clonal growth capability. The siRNA mediated HDAC8 knockdown inhibited clonogenic growth of UCCs (Figure 3B). The transfection of HDAC8 siRNA in VM-CUB1 and UM-UC-3 cells caused a moderate reduction of colony numbers compared to transfection of irrelevant siRNA by up to 30%. The relative size of the HDAC8 siRNA transfected colonies is reduced in 639-V in comparison to irrelevant siRNA. In VM-CUB1, SW-1710, RT-112 and UM-UC-3 cells the colony size remains constant between irrelevant control and HDAC8 siRNA transfection (data not shown).

Bottom Line: Efficient siRNA-mediated knockdown of HDAC8 reduced proliferation up to 45%.Expression of thymidylate synthase was partly reduced; PARP-cleavage was not detected.The influence of the pharmacological inhibition on clonogenic growth and migration show a cell line- and inhibitor-dependent reduction with the strongest effects after treatment with compound 5 and compound 6.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: Previous studies have shown that class-I histone deacetylase (HDAC) 8 mRNA is upregulated in urothelial cancer tissues and urothelial cancer cell lines compared to benign controls. Using urothelial cancer cell lines we evaluated whether specific targeting of HDAC8 might be a therapeutic option in bladder cancer treatment.

Methods: We conducted siRNA-mediated knockdown and specific pharmacological inhibition of HDAC8 with the three different inhibitors compound 2, compound 5, and compound 6 in several urothelial carcinoma cell lines with distinct HDAC8 expression profiles. Levels of HDAC and marker proteins were determined by western blot analysis and mRNA levels were measured by quantitative real-time PCR. Cellular effects of HDAC8 suppression were analyzed by ATP assay, flow cytometry, colony forming assay and migration assay.

Results: Efficient siRNA-mediated knockdown of HDAC8 reduced proliferation up to 45%. The HDAC8 specific inhibitors compound 5 and compound 6 significantly reduced viability of all urothelial cancer cell lines (IC₅₀ 9 - 21 μM). Flow cytometry revealed only a slight increase in the sub-G1 fraction indicating a limited induction of apoptosis. Expression of thymidylate synthase was partly reduced; PARP-cleavage was not detected. The influence of the pharmacological inhibition on clonogenic growth and migration show a cell line- and inhibitor-dependent reduction with the strongest effects after treatment with compound 5 and compound 6.

Conclusions: Deregulation of HDAC8 is frequent in urothelial cancer, but neither specific pharmacological inhibition nor siRNA-mediated knockdown of HDAC8 impaired viability of urothelial cancer cell lines in a therapeutic useful manner. Accordingly, HDAC8 on its own is not a promising drug target in bladder cancer.

Show MeSH
Related in: MedlinePlus